Conformation of the N-Terminal Segment of a Monocysteine Mutant of Troponin I from Cardiac Muscle

Abstract

A monocysteine mutant of cardiac muscle troponin I, cTnI(S5C/C81I/C98S), was generated from a mouse cTnI cDNA clone and expressed in a bacterial system. Cys-5 was modified with the fluorescent sulfhydryl reagent IAANS to probe the conformation of the N-terminal extension of the mutant and the mutant complexed with cardiac muscle troponin C. Our emphasis was on the effect of phosphorylation of Ser-23 and Ser-24 by protein kinase A on the conformation of the N-terminal segment. Phosphorylation resulted in an 8-nm red-shift of the emission spectrum of the attached IAANS probe and a reduction of its quantum yield by a factor of 4-5. The intensity decay of nonphosphorylated IAANS-labeled mutant was complex and had to be described by a sum of three exponential terms, with lifetimes in the range 0.1-5 ns. A fourth component in the range 7-9 ns was required to describe the intensity decay of the phosphorylated mutant. Phosphorylation also reduced the weighted mean lifetime, consistent with the changes observed in the steady-state fluorescence parameters and a 33% decrease in the global rotational correlation time calculated from anisotropy decay data. This change in correlation time suggested a decrease in the axial ratio of the protein. The fluorescence changes of the labeled mutant induced by phosphorylation were carried over to its complex with troponin C. The Stern-Volmer plots of acrylamide quenching of the steady-state fluorescence were essentially linear for nonphosphorylated mutant but displayed pronounced concave downward curvatures for the phosphorylated protein under all conditions studied. The present results are interpreted in terms of a more compact hydrodynamic shape of the phosphorylated cTnI mutant and are consistent with a folded conformation of the N-terminal extension induced by phosphorylation of the two serines. These conformational changes may play a role in the modulation of cardiac muscle contractility by troponin I phosphorylation.

Effects of PKA Phosphorylation of Cardiac Troponin I and Strong Crossbridge on Conformational Transitions of the N-Domain of Cardiac Troponin C in Regulated Thin Filaments

“…The structural consequence of phosphorylation of cTnI on protein-protein interactions that occur within the cTn complex as well as conformational changes of cTnI have been extensively studied using NMR (22,23,26,39,40) and FRET (21,33,(41)(42)(43). These studies suggest that the N-terminal extension of cTnI, most likely the residues immediately before the PKA phosphorylation sites, interact with the N-domain of cTnC.…”

Ca2+-induced Conformational Transition in the Inhibitory and Regulatory Regions of Cardiac Troponin I

“…In these cTnI mutants, the endogenous Trp 192 was changed to Phe and the two endogenous Cys 81 and Cys 98 were mutated to Ser and Ile, respectively. Construction of cTnI mutant clones, protein expression, purification, and characterization of the expressed proteins were as described (10). The single-cysteine cTnI mutants were labeled with 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid in the presence of 6 M urea (11).…”

Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance

“…pET-3d expression vectors (Novagen) containing mouse cardiac TnI, chicken slow TnC, and three single cysteine mutants (TnI132, TnI149, and TnC89) were kindly provided by the laboratory of Herbert C. Cheung. Details of these clones have been described elsewhere (17)(18)(19). The cardiac nomenclature for the TnC and TnI mutants is specified according to GenBank accession numbers A27204 (20) and AAA16157 (21), respectively.…”

“…The DNA was transformed into Escherichia coli BL21 (DE3) cells grown in Terrific Broth media (Difco). The recombinant proteins were isolated and purified according to established protocols (17,19,23).…”